It is known that droplets with a volume of more than 10 μl can be dispensed from the air very easily, since if the pipette is correctly manipulated, the droplets leave the pipette tip of their own accord. The droplet size is then determined by the physical properties of the sample liquid, such as surface tension or viscosity. The droplet size thus limits the resolution of the quantity of liquid to be dispensed.
The aspirating and dispensing, i.e. the pipetting of liquid samples with a volume of less than 10 μl, in contrast, typically requires instruments and techniques which guarantee the dispensing of such small samples. The dispensing of a liquid with a pipette tip, i.e. with the endpiece of a device for aspirating and/or dispensing sample liquid, can occur from the air (“from air”) or by touching a surface. This surface can be the solid surface of a container (“on tip touch”), into which the liquid sample is to be dispensed. It can also be the surface of a liquid in this container (“on liquid surface”). A mixing procedure following the dispensing is recommended—particularly for very small sample volumes in the nanoliter or even picoliter range—so that uniform distribution of the sample volume in a diluent is ensured.
Disposable tips significantly reduce the danger of unintentional transfer of parts of the sample (contamination). Simple disposable tips are known (so-called “air-displacement tips”), whose geometry and material is optimized for the exact aspirating and/or dispensing of very small volumes. The use of so-called “positive-displacement tips”, which have a pump plunger inside, is also known.
Systems for separating samples from a liquid are known as pipettors. Such systems serve, for example, for dispensing liquids into the wells of Standard Microtitration Plates™ (trademark of Beckman Coulter, Inc., 4300 N. Harbour Blvd., P.O. Box 3100 Fullerton, Calif., USA 92834) and/or microplates with 96 wells. The reduction of the sample volumes (e.g. for filling high-density microplates with 384, 864, 1536, or even more wells) plays an increasingly important role, with the precision of the sample volume dispensed being assigned a great importance. The elevation of the number of samples typically also requires miniaturization of the experiment, so that the use of a pipettor is necessary and special requirements must be placed on the precision of sample volumes and the accuracy of the movement control and/or of the dispensing of this pipettor.
The precision of a pipetting system is judged on the basis of the accuracy (ACC=accuracy) and reproducibility (CV=coefficient of variation) of the liquid samples dispensed. The reproducibility has greater significance assigned to it in this case, since systematic errors can, if necessary, be compensated by means of suitable correction parameters. In principle, two basic modes are differentiated in pipetting: single pipetting and multipipetting. In the single pipetting mode, a liquid sample is aspirated and dispensed at another location. In the multipipetting mode, a larger volume of liquid is aspirated at one time and subsequently dispensed in several—typically equivalent—portions (aliquots) at one or more different locations, e.g. in various wells of a Standard Microtitration Plate™.
Simpler pipettors, so-called “open systems”, connect the reservoir for the liquid to be pipetted with the pipette tip via a line into which a dispensing pump can be inserted. The dispensing pumps are typically implemented as piston pumps. For aspirating the sample, only the pump is set into operation, the pipette tip merely passively relays the liquid flow. For dispensing a sample volume, the pump is then switched off or bridged. For example, a pipette tip in the form of a microejection pump is known from European Patent No. 0 725 267, with which a liquid sample is actively separated. The supply of the liquid occurs due to the hydrostatic pressure obtaining in the line between the reservoir and the pipette tip. Such systems are relatively economical, but they have the disadvantage that the hydrostatic pressure can vary within a wide range. The reproducible dispensing of very small volumes in the nanoliter or even picoliter range is impaired thereby.
In addition, a “closed” system of this class is known from U.S. Pat. No. 5,927,547. In this system as well, only the pump is set in operation for aspirating; the pipette tip merely passively relays the liquid flow. In this system, however, the dispensing pump is put in operation—for generating a specific pressure in the tubing system to the pipette tip—for dispensing the liquid. For separating volumes in the picoliter to nanoliter range, piezoelectric driven tips and/or microejection pumps—as also known from European Patent No. 0 725 267—are used, in which the liquid or sample volumes are actively ejected out of the pipette tip. The pressure between the dispensing pump and the pipette tip is monitored with a sensor. The feed of the dispensing pump of this rather expensive and complex system is then regulated via a pressure sensor and an attached processing unit. Thus, synchronization between the microejection pump and the dispensing pump occurs.
The determination of the sample volume with a valve located in the immediate vicinity of the pipette tip or in the pipette tip itself, which, for example, is implemented as a solenoid or a piezovalve and is opened briefly, is also known. In this case, an initial pressure is used in the system. This opening is known in single action form or also in intervals. For dispensing a defined volume, the initial pressure must be adjusted very exactly to the liquid properties and the atmospheric conditions (above all to the air pressure). The effects influence each other strongly, so that the system must be readjusted upon a change of the liquid and/or its properties or the surrounding conditions.